Potentially pathogenic alterations have been identified in individuals with autism spectrum disorders (ASDs) within a variety of key neurodevelopment genes. While this hints at a common ASD molecular ...etiology, gaps persist in our understanding of the neurodevelopmental mechanisms impacted by genetic variants enriched in ASD patients. Induced pluripotent stem cells (iPSCs) can model neurodevelopment in vitro, permitting the characterization of pathogenic mechanisms that manifest during corticogenesis. Taking this approach, we examined the transcriptional differences between iPSC-derived cortical neurons from patients with idiopathic ASD and unaffected controls over a 135-day course of neuronal differentiation. Our data show ASD-specific misregulation of genes involved in neuronal differentiation, axon guidance, cell migration, DNA and RNA metabolism, and neural region patterning. Furthermore, functional analysis revealed defects in neuronal migration and electrophysiological activity, providing compelling support for the transcriptome analysis data. This study reveals important and functionally validated insights into common processes altered in early neuronal development and corticogenesis and may contribute to ASD pathogenesis.
The levels of methyl-CpG-binding protein 2 (MeCP2) are critical for normal post-natal development and function of the nervous system. Loss of function of MeCP2, a transcriptional regulator involved ...in chromatin remodeling, causes classic Rett syndrome (RTT) as well as other related conditions characterized by autism, learning disabilities, or mental retardation. Increased dosage of MeCP2 also leads to clinically similar neurological disorders and mental retardation. To identify molecular mechanisms capable of compensating for altered MeCP2 levels, we generated transgenic Drosophila overexpressing human MeCP2. We find that MeCP2 associates with chromatin and is phosphorylated at serine 423 in Drosophila, as is found in mammals. MeCP2 overexpression leads to anatomical (i.e., disorganized eyes, ectopic wing veins) and behavioral (i.e., motor dysfunction) abnormalities. We used a candidate gene approach to identify genes that are able to compensate for abnormal phenotypes caused by MeCP2 increased activity. These genetic modifiers include other chromatin remodeling genes (Additional sex combs, corto, osa, Sex combs on midleg, and trithorax), the kinase tricornered, the UBE3A target pebble, and Drosophila homologues of the MeCP2 physical interactors Sin3a, REST, and N-CoR. These findings demonstrate that anatomical and behavioral phenotypes caused by MeCP2 activity can be ameliorated by altering other factors that might be more amenable to manipulation than MeCP2 itself.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Copy number variations (CNVs) are a major cause of genetic disruption in the human genome with far more nucleotides being altered by duplications and deletions than by single nucleotide polymorphisms ...(SNPs). In the multifaceted etiology of autism spectrum disorders (ASDs), CNVs appear to contribute significantly to our understanding of the pathogenesis of this complex disease. A unique resource of 42 extended ASD families was genotyped for over 1 million SNPs to detect CNVs that may contribute to ASD susceptibility. Each family has at least one avuncular or cousin pair with ASD. Families were then evaluated for co-segregation of CNVs in ASD patients. We identified a total of five deletions and seven duplications in eleven families that co-segregated with ASD. Two of the CNVs overlap with regions on 7p21.3 and 15q24.1 that have been previously reported in ASD individuals and two additional CNVs on 3p26.3 and 12q24.32 occur near regions associated with schizophrenia. These findings provide further evidence for the involvement of ICA1 and NXPH1 on 7p21.3 in ASD susceptibility and highlight novel ASD candidates, including CHL1, FGFBP3 and POUF41. These studies highlight the power of using extended families for gene discovery in traits with a complex etiology.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The ATP-binding cassette, subfamily A (ABC1), member 7 (ABCA7) gene is associated with Alzheimer’s disease (AD) risk in populations of African, Asian, and European ancestry1-5. Numerous ABCA7 ...mutations contributing to risk have been identified, including a 44 base pair deletion (rs142076058) specific to individuals of African ancestry and predicted to cause a frameshift mutation (p.Arg578Alafs) (Cukier et al., 2016). The UMi043-A human induced pluripotent stem cell line was derived from an African American individual with AD who is heterozygous for this deletion and is a resource to further investigate ABCA7 and how this African-specific deletion may influence disease pathology.
The UMi028-A-2 human induced pluripotent stem cell line carries a homozygous mutation (rs377155188, C>G, p.S1038C) in the tetratricopeptide repeat domain 3 (TTC3) gene that was introduced via ...CRISPR/Cas9 genome editing. The line was originally derived from a neurologically normal male and has been thoroughly characterized following editing. The p.S1038C variant has been shown to potentially contribute to the risk of late onset Alzheimer’s disease and is a resource to further investigate the consequences of TTC3 and this alteration in disease pathology.
Despite significant progress in the genetics of autism spectrum disorder (ASD), how genetic mutations translate to the behavioral changes characteristic of ASD remains largely unknown. ASD affects ...1-2% of children and adults, and is characterized by deficits in verbal and non-verbal communication, and social interactions, as well as the presence of repetitive behaviors and/or stereotyped interests. ASD is clinically and etiologically heterogeneous, with a strong genetic component. Here, we present functional data from syngap1 and shank3 zebrafish loss-of-function models of ASD. SYNGAP1, a synaptic Ras GTPase activating protein, and SHANK3, a synaptic scaffolding protein, were chosen because of mounting evidence that haploinsufficiency in these genes is highly penetrant for ASD and intellectual disability (ID). Orthologs of both SYNGAP1 and SHANK3 are duplicated in the zebrafish genome and we find that all four transcripts (syngap1a, syngap1b, shank3a and shank3b) are expressed at the earliest stages of nervous system development with pronounced expression in the larval brain. Consistent with early expression of these genes, knockdown of syngap1b or shank3a cause common embryonic phenotypes including delayed mid- and hindbrain development, disruptions in motor behaviors that manifest as unproductive swim attempts, and spontaneous, seizure-like behaviors. Our findings indicate that both syngap1b and shank3a play novel roles in morphogenesis resulting in common brain and behavioral phenotypes.
A missense variant in the tetratricopeptide repeat domain 3 (TTC3) gene (rs377155188, p.S1038C, NM_003316.4:c 0.3113C>G) was found to segregate with disease in a multigenerational family with ...late-onset Alzheimer’s disease. This variant was introduced into induced pluripotent stem cells (iPSCs) derived from a cognitively intact individual using CRISPR genome editing, and the resulting isogenic pair of iPSC lines was differentiated into cortical neurons. Transcriptome analysis showed an enrichment for genes involved in axon guidance, regulation of actin cytoskeleton, and GABAergic synapse. Functional analysis showed that the TTC3 p.S1038C iPSC-derived neuronal progenitor cells had altered 3-dimensional morphology and increased migration, while the corresponding neurons had longer neurites, increased branch points, and altered expression levels of synaptic proteins. Pharmacological treatment with small molecules that target the actin cytoskeleton could revert many of these cellular phenotypes, suggesting a central role for actin in mediating the cellular phenotypes associated with the TTC3 p.S1038C variant.
GABAergic synaptic transmission is known to play a critical role in the assembly of neuronal circuits during development and is responsible for maintaining the balance between excitatory and ...inhibitory signaling in the brain during maturation into adulthood. Importantly, defects in GABAergic neuronal function and signaling have been linked to a number of neurological diseases, including autism spectrum disorders, schizophrenia, and epilepsy. With patient-specific induced pluripotent stem cell (iPSC)-based models of neurological disease, it is now possible to investigate the disease mechanisms that underlie deficits in GABAergic function in affected human neurons. To that end, tools that enable the labeling and purification of viable GABAergic neurons from human pluripotent stem cells would be of great value.
To address the need for tools that facilitate the identification and isolation of viable GABAergic neurons from the in vitro differentiation of iPSC lines, a cell type-specific promoter-driven fluorescent reporter construct was developed that utilizes the human vesicular GABA transporter (hVGAT) promoter to drive the expression of mCherry specifically in VGAT-expressing neurons. The transduction of iPSC-derived forebrain neuronal cultures with the hVGAT promoter-mCherry lentiviral reporter construct specifically labeled GABAergic neurons. Immunocytochemical analysis of hVGAT-mCherry expression cells showed significant co-labeling with the GABAergic neuronal markers for endogenous VGAT, GABA, and GAD67. Expression of mCherry from the VGAT promoter showed expression in several cortical interneuron subtypes to similar levels. In addition, an effective and reproducible protocol was developed to facilitate the fluorescent activated cell sorting (FACS)-mediated purification of high yields of viable VGAT-positive cells.
These studies demonstrate the utility of the hVGAT-mCherry reporter construct as an effective tool for studying GABAergic neurons differentiated in vitro from human pluripotent stem cells. This approach could provide a means of obtaining large quantities of viable GABAergic neurons derived from disease-specific hiPSCs that could be used for functional assays or high-throughput screening of small molecule libraries.
•Development of human VGAT promoter–reporter lentiviral construct•Expression of mCherry from the VGAT promoter was specific to GABAergic neurons.•Effective labeling of several different GABAergic interneuron subtypes•High yields of viable GABAergic neurons using novel fluorescent activated cell sorting (FACS)
Background
We identified a rare, nonsynonymous variant in the tetratricopeptide repeat domain 3 (TTC3) gene that segregated in all 11 Alzheimer disease (AD) individuals in a non‐Hispanic white late ...onset Alzheimer disease (LOAD) family (mean AAO=75.7 years, Kohli, et al, 2016). This missense alteration, rs377155188 (p.S1038C), is predicted to be deleterious by five in silico algorithms and extremely rare in the gnomAD database (allele frequency=3.231x10‐5). Studies have reported that cortical TTC3 expression is reduced in LOAD patients and negatively correlated with the AD neuropathology.
Method
To understand the mechanism by which the TTC3 alteration may contribute to LOAD risk, CRISPR/Cas9 genome edited induced pluripotent stem cells (iPSCs) were developed to examine cellular consequences in neuronal cells, which can otherwise only be collected postmortem. Starting with an iPSC line derived from a healthy 45 year old male donor of European ancestry, ASE‐9203, genome editing was performed by introducing an sgRNA cloned into the Cas9 containing TLCV2 plasmid (Addgene) simultaneously with a ssODN template.
Result
An independent homozygous edited iPSC clone was isolated and verified by Sanger sequencing to carry the TTC3 variant, thus creating an isogenic pair of iPSCs. The stem cells were differentiated into forebrain neurons for 70 days and evaluated. Quantitative PCR analysis demonstrated that TTC3 levels were decreased in edited compared to unedited iPSCs, as well as day 30 and day 70 differentiated neurons. Since there is evidence that modulation of TTC3 affects neurite growth (Berto, et al, 2007), morphological measures of axon formation were assessed in differentiating neuronal cultures using the Incucyte Zoom. Preliminary studies demonstrate an increase in neurite outgrowth, which phenotypically corresponds with previous studies of a decrease in TTC3 function. Levels of amyloid beta 40 and 42 released from day 65 edited and unedited neurons were not significantly different.
Conclusion
Results suggest that the TTC3 p.S1038C variant causes a loss of function. Utilizing a CRISPR genome edited iPSC carrying a homozygous alteration in TTC3 will enable us to determine how this genetic alteration which may contribute to specific cellular phenotypes and, on a broader scale, LOAD risk.
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